Blood banking centers fractionate blood into various components such as plasma, erythrocytes, plateletes, and leukocytes. Each of these components is then used for a specific application. The blood cell differential in whole blood is described in A. K. Abbas et al, "Introduction to Immunology", Cellular and Molecular Immunology, p. 14, Harcourt Brace Jovanovich, Inc. (1991).
Current methods for isolating erythrocytes, platelets and leukocytes from whole blood include using low-speed centrifugation. According to this method, erythrocytes and platelets are separated from leukocytes based on their density. A top layer containing plasma and platelets is removed for the preparation of plasma and platelets and the middle layer containing leukocytes (buffy coats) may be used for induction of cytokines, such as interferons, for isolation of specific cell types for the study of the immune system or for immunotherapy. The bottom layer which contains erythrocyte-rich red cells may be used in transfusions.
This method of isolating red blood cells or platelets from leukocytes by centrifugation is tedious and often does not result in complete separation of the blood cell types. As a result, blood bank centers have begun to use various filtration units to remove leukocytes from the desired red blood cell or platelet preparations. Examples of these filtration units include the Pall RCM.RTM. system of Pall Corporation (East Hills, N.Y.), Leukotrap.RTM. RC/PL system of Miles Pharmaceuticals, the LeukoNet.RTM. system of HemaSure, Inc. and the Sepacell-R/PL.TM. system of Baxter Healthcare Corporation. These are online filters which collect blood directly from the human vein, rather than indirectly from the centrifugation system. Such online filter units trap and separate leukocytes from erythrocytes and platelets based on cell size exclusion. The smaller erythrocytes easily pass through the filter while the larger leukocytes remained behind trapped in the filter. These filter systems result in a reduction of leukocytes ranging from 82.2% to 99%, depending on the filter system used. [Beitr Infusionsther, 30:152-156 (1992)]
Typically, filters used to prepare red-blood cell or platelet-rich preparations are discarded. However, prior methods have been reported in which leukocytes which remain trapped behind in the filter units have been recovered. For example, one method involves back-flushing five times with 50 mL phosphate buffered saline ("PBS") at 4.degree. C. using a 60 mL syringe [R. Longley et al, J. Immuno. Methods, 121:33-38 (1989)]. The "back-flushing" involves flushing the filters in one direction, opposite to the one which is initially used to remove the leukocytes from the desired preparation. The recovered leukocytes at this point in the purification process, still contain a substantial amount of red blood cells. In order to remove the red blood cells, the eluted leukocyte preparations are then further purified by Histopaque gradient centrifugation. A protocol for the isolation of mononuclear cells by Ficoll-Hypaque gradient centrifugation ("histopaque") is described in J. E. Coligan, Current Protocols in Immunology, Section I, Units 7.1-7.2, John Wiley & Sons (1994). The mononuclear cells are collected in the interface layer and washed with PBS. Typically, a total of 2.76+/-1.17 .times.10.sup.8 leukocytes per filter, having a viability of more than 95%, will be isolated by this two-step process. In a 5-10 ml sample, where one would expect 2.times.10.sup.9 leukocytes to be present per filter, this represents a 14% recovery of leukocytes. In comparison to some published methods, where a 70% recovery of buffy coats is generally anticipated, this recovery rate is small. Leukocytes isolated in such a manner have been shown to be functional in response to phytohemagglutinin (PHA), conconavalin (CONA), and pokeweed mitogens (PWM).
The elution of leukocytes from filters is also described in Kuroda et al, U.S. Pat. No. 4,416,777. This method also required a multi-step process to facilitate removal of red blood cells from leukocytes using a variety of elution solutions: PBS in combination with polyvinyl-pyriolidone, sodium casein, polyvinyl-alcohol or gelatin.
In view of the importance of purifying fully functional human leukocytes for the manufacture of therapeutic products and other applications, many attempts have been made to retrieve leukocytes from various sources. However, methods used to date continue to have many drawbacks. For example, the methods of Kuroda et al, U.S. Pat. No. 4,416,777 and Longley et al, supra, are both multi-step and, therefore, tedious processes. The published methods produce a relatively low number of leukocytes per filter unit. In addition, known methods require processing of the filters within 24 hours after blood collection; otherwise the viability of the leukocytes is greatly impaired. As a result, improved methods for retrieving leukocytes from recycled blood product preparation filters continue to be needed.